Core Hemoglobinopathy Laboratory We propose to develop a Core Hemoglobinopathy Laboratory within the Northern California Sickle Cell Center. The Core will have two units: a Diagnostic and Functional Studies unit and a Structural Studies unit. The specific aims of this core laboratory are: 1) To provide essential hemoglobin related laboratory services for patients entered in clinical studies. 2) To facilitate identification of hemoglobin variants and determine their functional significance. 3) To develop novel mass spectrometric techniques to identify hemoglobin variants. 4) To develop a national resource available to the entire sickle cell and hematology community. The Diagnostic and Functional Studies laboratory will provide essential hemoglobin laboratory analysis for all clinical research studies, perform functional characterization of hemoglobins, will coordinate specimen collection and distribution to other components of the center and data collection for clinical research studies. The following tests will be offered: hemoglobin phenotype, quantitative Hb A and S, Hb A2 and Hb F levels, F-reticulocyte and F-cell counts, pitted red cells counts, hemoglobin oxygen affinity, hemoglobin stability, minimum gelling concentration and solubility. Availability of both structural and functional analysis of abnormal hemoglobins will be particularly useful in characterizing compound heterozygotes with HbS. The Structural Studies laboratory will provide structural characterization of variant human hemoglobins as well as the hemoglobins generated by Dr. Rubin in his transgenic mice experiments. We estimate that over 1000 variant hemoglobins from our center, other sickle cell centers and other participating institutes (including variants detected during newborn screening) will be analyzed per year. Priority will be given to samples from symptomatic patients and where information is essential for genetic counseling. We propose to use electrospray ionization mass spectrometry (ESI MS) as a primary technique in detection and identification of hemoglobin variants, including "electrophoretically silent" variants that differ in mass but not charge from normal hemoglobins. Individual blood hemolysates are passed into the mass spectrometer every few minutes and the molecular weights and amounts of the globin chains present are obtained. Further variant characterization requires protein digestion followed by mass analysis of proteolytic fragments and MS/MS peptide sequencing, the latter required to solve ambiguous cases. During the tenure of this grant we will investigate the possibility: (1) of automating the basic ESI MS for hemolysate analysis to allow large throughput of samples, (2) of an on-line separation/mass analysis of the intact globin chains (HPLC/MS or capillary zone electrophoresis CZE/MS), (3) of micro LC/MS/MS or CZE/MS/MS for simultaneous separation, mass determination and sequencing of proteolytic fragments, (4) of fingerprinting variant hemoglobins by collision induced dissociation (CID) of intact globin chains. We also intend to develop the mass spectrometric data system to provide automatic provisional identification of amino acid changes and possible variant identify.